The long term objective of this proposal is to define the mechanisms which control intracellular pH (pHi) and cell volume of colonic epithelial cells during changes in transepithelial ion transport. The two specific topics of interest are the diarrheal secretion of ions and water due to activation of second messengers pathways, and the stimulation of NaCl and water absorption by pro-absorptive compounds normally found in the colon (short-chain fatty acids: SCFAs). Therefore our studies relate to understanding cell homeostatic mechanisms during diarrhea, and in the presence of agents which oppose diarrheal disorders. Our experimental system is a human tissue culture model (HT29/Ci cells) which preliminary studies identify as a model of a colonic ion-transporting epithelial cell. The cloned HT29/Ci cell responds at a cellular and epithelial level to both second messengers and SCFAs such that it will be useful for studies of both secretory and absorptive functions of the intestinal epithelia. Increases in cAMP cause C1- secretion in the colon and in the HT29/Ci cell. SCFAs cause increased NaCl absorption in the colon and stimulate apical Na+ transport pathways in the HT29/Ci cell. Specific experiments will measure pHi, cell volume, and transcellular (Na+, K+, Cl-) transport in HT29/Cl cells, in order to define the integrated response of the cell with respect to these parameters. The first aim is to identify which second messenger systems cause alterations in cell pH and volume homeostasis. Next the membrane mechanisms affected by the different second messenger systems will be identified and comparisons made between the effects of different second messenger systems. Finally, experiments will examine whether the observed effects of different second messengers on transepithelial transport may be dissociated from those on pHi or volume. Studies of SCFAs will first identify and characterize the routes of SCFAs flux, since this is not well established in colonic cells. Subsequent experiments will define the transport pathways activated by SCFAs to change cellular pHi or volume. Studies of SCFAs will first identify and characterize the routes of SCFA flux, since this is not well established in colonic cells. Subsequent experiments will define the transport pathways activated by SCFAs to change cellular pHi and volume, and transcellular ion transport.